Research Article 2743

IntroductionThe nucleolus is the most conspicuous nuclear compartment whereseveral steps in ribosome biogenesis take place: transcription ofthe 5.8S, 18S and 28S rRNA genes, processing of the 47S pre-rRNA and assembly of pre-ribosomal particles (Hadjiolov, 1985).There are ≥300 copies of rRNA genes in the human genome(McConkey and Hopkins, 1964; Jeanteur and Attardi, 1969; Stultset al., 2008) located in the nucleolar organizer regions (NORs) onthe short arms of five pairs of acrocentric chromosomes (Hendersonet al., 1972; Hadjiolov, 1985). The number of rDNA repeats inindividual human NORs is in the range of 1 to ≥140 (Stults et al.,2008). The levels of rRNA gene transcription vary in different celltypes depending on cell proliferation rates and the demand forrRNA. Short-term regulation of rRNA production occurspredominantly through modulation of rDNA transcription rates,whereas long-term regulation also involves changes in the numberof transcribed rRNA gene copies (Schwarzacher and Wachtler,1983; McStay and Grummt, 2008). Only 20–50% of all rRNAgenes are transcriptionally active in most human cells (Miller andBakken, 1972; Gagnon-Kugler et al., 2009). They are believed toexist in a nucleosome-free state, whereas the silent copies arepacked as regular chromatin (Hamkalo and Miller, 1973; Sogo andThoma, 2004). Although the precise organization of active andrepressed ribosomal genes within human NORs is unknown, it isclear that some NORs are composed entirely of inactive rRNAgenes (silent NORs), whereas other NORs (competent NORs) hostactive only, or a mixture of active and inactive, rRNA genes (deCapoa et al., 1988). The number of competent NORs varies amongcell types and even within the same cell type in each individual;typically, more than half (50–100%) of all NORs in rapidly growingcells are competent (Mikelsaar et al., 1977; Miller et al., 1977;Mikelsaar and Schwarzacher, 1978; Schmiady et al., 1979;

Zakharov et al., 1982; Heliot et al., 2000; Kalmarova et al., 2007).During interphase, all competent NORs congregate within one, ora few, nucleoli per nucleus (Anastassova-Kristeva, 1977;Schwarzacher and Wachtler, 1983; Hadjiolov, 1985). Even thoughthe organization and integrity of nucleoli depend on ongoing rRNAsynthesis, processing and transport (Hernandez-Verdun et al., 2002),localization to the nucleolar compartment(s) is not restricted totranscriptionally active loci. Thus, many non-rDNA loci (vanKoningsbruggen et al., 2010; Puvion-Dutilleul et al., 1991; Nemethet al., 2010), as well as some, or all, silent NORs (D.S.D.,unpublished) (Sullivan et al., 2001; Kalmarova et al., 2007) alsoassociate with nucleoli.

Despite the fact that the discovery of the nucleolus dates backto the late 18th century and early 19th century (Schwarzacher andWachtler, 1983; Mosgoeller, 2004), and the thousands ofpublications resulting from many decades of intensive research,consensus about its structural and functional organization has notbeen reached (Olson, 2004). Although not as hotly debated as thelocation of rRNA gene transcription, rDNA replication is alsoassociated with unresolved issues regarding both the location ofreplicating ribosomal genes and the usage of specific replicationorigins. Attempts to map replication origin positions in the humanrDNA loci have generated conflicting results (see the Results andDiscussion sections). Furthermore, biochemical studies haverevealed that, in mammals, rDNA replicates throughout S phase(Balazs and Schildkraut, 1971; Epner et al., 1981; Berger et al.,1997; Li et al., 2005). It is considered that the transcriptionallyactive rDNA copies replicate in early S phase, whereas the silentcopies replicate in late S phase (Li et al., 2005). However,immunofluorescence microscopy methodologies for visualizationof nascent rDNA failed to detect replicative activity inside nucleolibefore mid S phase (Junera et al., 1995; Pliss et al., 2005). The

SummaryTypically, only a fraction of the ≥600 ribosomal RNA (rRNA) gene copies in human cells are transcriptionally active. Expressed rRNAgenes coalesce in specialized nuclear compartments – the nucleoli – and are believed to replicate during the first half of S phase.Paradoxically, attempts to visualize replicating rDNA during early S phase have failed. Here, I show that, in human (HeLa) cells, early-replicating rDNA is detectable at the nucleolar periphery and, more rarely, even outside nucleoli. Early-replicated rDNA relocates tothe nucleolar interior and reassociates with the transcription factor UBF, implying that it predominantly represents expressed rDNAunits. Contrary to the established model for active gene loci, replication initiates randomly throughout the early-replicating rDNA. Bycontrast, mostly silent rDNA copies replicate inside the nucleoli during mid and late S phase. At this stage, replication origins are firedpreferentially within the non-transcribed intergenic spacers (NTSs), and ongoing rDNA transcription is required to maintain thisspecific initiation pattern. I propose that the unexpected spatial dynamics of the early-replicating rDNA repeats serve to ensurestreamlined efficient replication of the most heavily transcribed genomic loci while simultaneously reducing the risk of chromosomebreaks and rDNA hyper-recombination.

Key words: Gene expression, Human rRNA genes, Nucleolus, Replication factory, Replication origin

Accepted 26 April 2011Journal of Cell Science 124, 2743-2752 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.082230

DNA replication initiation patterns and spatialdynamics of the human ribosomal RNA gene lociDaniela S. DimitrovaThe Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UKdaniela.dimitrova@bbsrc.ac.uk

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goal of the present work was to investigate the location of theelusive early-replicating rDNA. My findings reveal complex,spatially and temporally differentiated regulation of the replicationof active and repressed rRNA genes in the course of S phase,which is possibly relevant to efficient rDNA replication andmaintenance of rDNA integrity.

ResultsrDNA replication occurs at the periphery of, or outside,nucleoli during early S phaseThe expressed rRNA genes are located and transcribed in thenucleolar interior. However, all descriptions of the distribution ofDNA replication sites during the first half of S phase state thatreplication takes place throughout the cell nucleus except insidenucleoli (Nakamura et al., 1986; Nakayasu and Berezney, 1989;van Dierendonck et al., 1989; Banfalvi et al., 1990; Humbert andUsson, 1992; O’Keefe et al., 1992; Hozak et al., 1993; Dimitrovaand Berezney, 2002). This is illustrated in Fig. 1 and supplementarymaterial Fig. S1. HeLa cells were incubated for 10 minutes with5-ethynyluridine (EU) and 5-bromo-2�-deoxyuridine (BrdU) tolabel nascent RNA and DNA, respectively. The distribution oftranscription (EU) foci is similar in all nuclei, whereas replication(BrdU) foci are arranged in different patterns typical for early (~0–5 hours into S phase, Fig. 1A–D), middle (~6–8 hours into Sphase, Fig. 1E–G) and late (~9–10 hours into S phase, Fig. 1H) Sphase. Nearly a quarter of the total nascent RNA output in HeLacells is produced by RNA polymerase I (PolI), as measured bynuclear RNA run-on assay (supplementary material Fig. S2),accounting for the strong fluorescence of the nascent rRNA foci.There was no detectable replicative activity inside nucleoli duringthe first half of S phase, and they appeared as hollow gaps in theearly S phase replication pattern (supplementary material Fig.S1A). When transcription and replication sites are visualizedsimultaneously, these gaps were filled with extremely bright nascentrRNA foci (Fig. 1A–D; supplementary material Fig. S1B). Bycontrast, several replication foci interspersed among the EU fociwere easily detectable in the nucleolar interior throughout mid andlate S phase (Fig. 1E–G), with the exception of the final 1–2 hoursof S phase (Fig. 1H). These observations raise a puzzling paradox:although light-microscopy experiments fail to detect replicationactivity inside nucleoli during the first half of S phase (Fig. 1;supplementary material Fig. S1) (Nakamura et al., 1986; Nakayasuand Berezney, 1989; O’Keefe et al., 1992; Junera et al., 1995;

Dimitrova and Gilbert, 1999; Pliss et al., 2005), biochemicalexperiments show that roughly half of all rDNA repeats arereplicated at this same time in mammalian cells (Balazs andSchildkraut, 1971; Epner et al., 1981; Berger et al., 1997; Li et al.,2005).

I reasoned that perhaps the solution to this paradox is to look forthe early-replicating ribosomal genes not inside, but around thenucleoli. To test this hypothesis, I applied three-dimensional DNAfluorescence in situ hybridization (3D DNA FISH) to detect rDNAin HeLa cells pulse-labeled for 10 minutes with 5-ethynyl-2�-deoxyuridine (EdU) to visualize active replication sites. The relativelysmall size of the fluorescent azide molecules used for EdU detectionallows them to penetrate dense nuclear structures, which might beinaccessible to bulky immunological reagents. For the same reason,fluorescent azide derivatives can react with EdU within double-stranded DNA, eliminating the necessity to use harsh procedures forDNA denaturation and thus ensuring superior chromatin structurepreservation – an important advantage for this particular study. Toexclude the possibility of artifactual rDNA redistribution caused byheat-induced chromatin spreading or breakage, I also used a gentleDNA FISH methodology, which avoids thermal denaturation ofDNA (van Dekken et al., 1988). The rDNA FISH signals appearedas several intense spots of various sizes, interspersed with morediffuse and less intense fluorescent signals (Figs 2–4). To visualizethe nucleolar compartment(s), the cells were stained with antibodiesspecific for the ubiquitous protein nucleolin. Owing to its diversefunctions in rRNA production and ribosome assembly (Mongelardand Bouvet, 2007), nucleolin levels are high in nucleoli (Spector etal., 1984; Rickards et al., 2007; Ugrinova et al., 2007), and nucleolinstaining is often used as a marker for the nucleolar territories (vanKoningsbruggen et al., 2010; Alcalay et al., 2005; Olson and Dundr,2005; Ma et al., 2007; Amin et al., 2008). Accordingly, all clustersof strongly fluorescent nascent rRNA foci in HeLa cells wereembedded within nuclear areas positive for nucleolin immunostaining(supplementary material Fig. S3). Simultaneous immunodetectionof the PolI transcription factor, upstream binding factor (UBF) wasperformed to identify transcriptionally active ribosomal gene copies(Mosgoeller et al., 1998), given that virtually all UBF staining wasclosely associated with bright EU foci (i.e. nascent rRNA) duringinterphase in HeLa cells (supplementary material Fig. S4). Consistentwith the results of the EU + BrdU labeling experiments (Fig. 1A–H), the three-dimensional confocal analysis of HeLa nuclei revealedthat rDNA FISH signals located inside the nucleolin-stained

2744 Journal of Cell Science 124 (16)

Fig. 1. Spatial organization of PolI transcription foci andDNA replication foci in HeLa cell nuclei during the courseof S phase. EU-labeled nascent RNA (red) was stained withAlexa-Fluor-594–azide, and BrdU-labeled nascent DNA(green) was detected with mouse anti-BrdU antibodies plusAlexa-Fluor-488-conjugated anti-mouse-IgG antibodies. Inthe main images, fluorescence intensity is optimized forvisualization of the strong nucleolar EU signals. The insetsshow brighter images where the nucleoplasmic transcriptionfoci are also visible, however, details in the nucleolar areasare lost. (A–D) Four consecutive confocal z-sections througha single nucleus in early S phase. Replication foci areexcluded from the nucleoli (the areas with clusters of brightfoci of EU-labeled rRNA). (E–H) Single confocal z-sectionsthrough four HeLa nuclei. Replication foci interspersed withtranscription foci inside nucleoli are detectable in nuclei inmid S phase (E–G), but rarely in late S phase (H). Scale bars:2 m.

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compartments did not colocalize with EdU in nuclei exhibiting theearly S-phase replication pattern (Fig. 2A; supplementary materialFig. S5A; Table 1). Notably, in the majority of early S nuclei, rDNAFISH signals overlapping EdU foci could be found at the peripheryof or, more rarely, even outside the nucleolar areas (Fig. 2B,supplementary material Fig. S5A; Table 1). The number of rDNAfoci engaged in DNA replication was low at any given moment (oneto a few), which could explain why the early-replicating perinucleolarrDNA foci have hitherto eluded detection. Even though both EdUstaining (D.S.D., unpublished) and rDNA FISH (D.S.D., unpublished)(Schofer et al., 1998; Pasero et al., 2002; Lebofsky and Bensimon,

2005) are sufficiently sensitive to detect individual extended DNAfibers, it is not possible to exclude that, for unknown reasons, rDNApotentially replicating inside the nucleolar interior during early Sphase cannot be detected by light microscopy. However, the positiveidentification reported here suggests that at least some of the earlyrDNA replication takes place at the edge of, or outside, nucleoli.

In contrast to early S phase, one or more replicating rDNAdomains (i.e. overlapping with EdU) were readily detectable inthe nucleolar interior during mid S phase (Fig. 3A; supplementarymaterial Fig. S5B; Table 1), but rarely in late S phase (Fig. 3B;supplementary material Fig. S5C; Table 1), in agreement with

2745Dynamics of human rDNA replication

Fig. 2. rRNA genes replicate at the nucleolarperiphery during early S phase. HeLa cells werepulse-labeled for 10 minutes with EdU andimmediately fixed. 3D DNA immunoFISH wasperformed to visualize rDNA (green), DNAreplicated during the EdU pulse (red), nucleolin(left-hand column, blue) and UBF (right-handcolumn, blue). A single confocal z-section (S4)from a nucleus exhibiting an early S-phasereplication pattern is shown. Lines passing throughtwo nucleoli were drawn across the images and theline-scan plots of fluorescence intensity versusdistance recorded along the lines are shown beloweach image. The three-dimensional confocal imagestack for this nucleus is shown in supplementarymaterial Fig. S5A. Small areas of interest (framed)are shown at higher magnification alongside therespective image (as RGB, as well as theirrespective red, green and blue image components).The nuclear areas stained with the anti-nucleolinantibodies (i.e. the nucleoli) are outlined withwhite contours in the confocal images and arehighlighted in a light blue color in the line-scanprofiles. rDNA FISH signals are classified as:intranucleolar, when enclosed within the contours;located at the nucleolar periphery, when straddlingor touching the contours from the outside; ordistant from nucleoli, when separated from thecontoured areas by nuclear space negative fornucleolin staining. Replicating rDNA (arrows)does not colocalize with UBF and is detected at thenucleolar periphery (B), but not inside nucleoli (A)in the early S-phase nucleus. Scale bars: 2 m.

Table 1. Proportions of HeLa cells exhibiting rDNA replication inside, at the periphery, or outside nucleoli at different stages ofS phase

Cells with rDNA Cells with rDNA Cells with rDNA Cells with no Number of replicating inside replicating at the replicating outside detectable replicating

S-phase stage cells analyzed nucleoli (%) nucleolar periphery (%) nucleoli (%) rDNA (%)

Early 128 0 (n=0) 53.1 (n=68) 12.5 (n=16) 34.4 (n=44)Mid 156 41.0 (n=64) 43.6 (n=68) 12.8 (n=20) 2.6 (n=4)Late 52 7.7 (n=4) 30.8 (n=16) 38.5 (n=20) 23.1 (n=12)

The scores are from a single experiment. Similar results were obtained in three independent experiments.

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previous reports (Junera et al., 1995; Pliss et al., 2005). In afraction of nuclei in mid and late S phase, colocalization ofrDNA with EdU was also detectable at the nucleolar peripheryand outside nucleoli (Table 1). Most intranucleolar replicationfoci did not contain rDNA (supplementary material Fig. S5B),consistent with the view that only a relatively small proportionof the DNA inside nucleoli represents rDNA (Puvion-Dutilleul etal., 1991).

Altered replication timing in cancer cells has been reported forindividual genomic loci (Dotan et al., 2008). Hence, it waspossible that, despite the general correlation between active geneexpression and early replication timing, as well as the reportedearly replication of expressed ribosomal genes in immortalizedmouse 3T3 fibroblasts (Li et al., 2005), the active rRNA genesmight be late-replicating in the transformed HeLa cells.Unfortunately, the UBF staining was not useful for revealing theexpression status of the early-replicating peri- or extra-nucleolar,or the late-replicating intranucleolar rDNA units, because they,and replication foci in general, did not overlap significantly withUBF (Figs 2 and 3, UBF). It is possible that the passage of thereplication machinery displaces UBF from DNA.

Replicated rDNA relocates to the nucleolar interior andreassociates with UBFTo overcome this difficulty, I performed a pulse-chase experiment toexamine UBF reassociation with previously replicated rDNA.Aliquots of HeLa cells pulse-labeled for 10 minutes with EdU (fromthe same cell population used in Figs 2 and 3) were transferred toEdU-free medium. At hourly intervals thereafter, aliquots of cellswere processed for UBF, nucleolin and rDNA immunoFISHcombined with EdU detection. The results from a 2-hour chase areshown in Fig. 4 and supplementary material Fig. S6. As shownpreviously (Manders et al., 1992; Dimitrova and Gilbert, 1999), thistime is sufficient for all DNA contained within a replication focus tocomplete replication. In striking contrast to the ‘real-time snapshots’during ongoing replication (Fig. 2A,B), EdU-positive rDNA FISHsignals were detected inside nucleoli in a large fraction (59.6%,n=146 cells) of the nuclei exhibiting an early-S-phase replicationpattern (Fig. 4A, nucleolin; supplementary material Fig. S6A). UBFcolocalized with the EdU-tagged rDNA in these cells (Fig. 4A,UBF), implying that the latter predominantly represents expressedrRNA genes. In nuclei exhibiting replication patterns from the secondhalf of S phase, rDNA FISH signals did not show substantial overlap

2746 Journal of Cell Science 124 (16)

Fig. 3. Location of replicating rDNA during midand late S phase. HeLa cells were pulse-labeledfor 10 minutes with EdU and immediately fixed.3D DNA immunoFISH was performed to visualizerDNA (green), DNA replicated during the EdUpulse (red), nucleolin (left-hand column, blue) andUBF (right-hand column, blue). Representativesingle confocal z-sections from nuclei exhibitingmid S-phase (A) or late S-phase (B) replicationpatterns are shown. Line-scan plots of fluorescenceintensity versus distance recorded along the yellowlines drawn across the images are shown beloweach image. The three-dimensional confocal imagestacks for each nucleus are shown insupplementary material Fig. S5B,C. Small areas ofinterest (framed) are shown at highermagnification alongside the respective image (asRGB, as well as their respective red, green andblue image components). The nuclear areas stainedwith the anti-nucleolin antibodies (i.e. the nucleoli)are outlined with white contours in the confocalimages and are highlighted in a light blue color inthe line-scan profiles. Replicating rDNA is locatedboth inside nucleoli and at the nucleolar periphery.Scale bars: 2 m.

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with UBF-stained regions after the chase (Fig. 4B, UBF), indicatingthat, similar to non-transformed cells, the silent rDNA repeatsreplicate later in S phase in HeLa cells. Similar results were obtainedin two more experiments using different populations of HeLa cells.Taken together, these findings suggest that expressed ribosomalgenes reposition to the edge of or outside nucleoli before theirreplication and, once replicated, shift back to the nucleolar interiorwhere they reassociate with UBF and become transcriptionallyactive. Importantly, the fact that silent rDNA repeats and other DNAare replicated inside nucleoli (Fig. 1E–G; Fig. 3A; supplementarymaterial Fig. S5B) indicates that the densely packed nucleolar interioris not prohibitive for assembly of functional replication factories orfor light-microscopy detection of replicating rDNA. Therefore, thesurprising spatial dynamics of the expressed ribosomal genes,whereby they engage in replication factories at the nucleolarperiphery, might be related to their transcriptional status.

The distribution of replication origin sites in the humanrDNA repeats differs from the established model for activegene lociReplication initiation sites tend to be excluded from transcribedsegments of the chromatin fiber (Hyrien et al., 1995; Mesner and

Hamlin, 2005; Dimitrova, 2006), and ongoing transcription isessential to sustain this preferential origin positioning (Dimitrova,2006). Previous studies of the distribution of replication origins inthe human rDNA clusters have generated disparate results(summarized in supplementary material Fig. S7) (Little et al.,1993; Yoon et al., 1995; Gencheva et al., 1996; Lebofsky andBensimon, 2005). The majority of studies (Yoon et al., 1995;Gencheva et al., 1996; Lebofsky and Bensimon, 2005) have madeno attempt to look for potential differences between early- andlate-replicating ribosomal gene units, whereas those that haveaddressed this issue (Little et al., 1993) have found no significantdifferences. To gain further insight into the distinct behavior ofexpressed and silent rRNA genes, I examined their replicationinitiation patterns by taking advantage of their different replicationtiming. Abrogation of the replication checkpoint in cells grown inthe presence of aphidicolin (Aph) leads to disassembly of existingreplication forks stalled by Aph and progressive activation of later-firing origins in the absence of actual genome replication(Dimitrova and Gilbert, 2000). Importantly, although checkpointinhibitors prolong S phase, neither the temporal order of repliconactivation nor the normal distribution of replication origins arealtered (Dimitrova and Gilbert, 2000). Therefore, this approach

2747Dynamics of human rDNA replication

Fig. 4. Inside nucleoli, UBF associates selectivelywith human rRNA genes replicated during earlyS phase but not mid S phase. HeLa cells werepulse-labeled with EdU, transferred to free mediumand fixed and processed 2 hours later. 3D DNAimmunoFISH was performed to visualize rDNA(green), DNA replicated during the EdU pulse (red),nucleolin (left-hand column, blue) and UBF (right-hand column, blue). Representative individualconfocal z-sections from nuclei exhibiting (A) anearly S-phase or (B) a mid S-phase replicationpattern are shown. Arrows point to EdU-overlapping rDNA FISH signals. Line-scan plots offluorescence intensity versus distance recordedalong the yellow lines drawn across the images areshown below each image. The three-dimensionalconfocal image stacks for each nucleus are shown insupplementary material Fig. S6. Small areas ofinterest (framed) are shown at higher magnificationalongside the respective image (as RGB, as well astheir respective red, green and blue imagecomponents). The nuclear areas stained with theanti-nucleolin antibodies (i.e. the nucleoli) areoutlined with white contours in the confocal imagesand are highlighted in a light blue color in the line-scan profiles. Scale bars: 2 m.

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can be used to map replication origin locations throughout S phase(see supplementary material Fig. S8 for further details on themethodology). I performed radioactive nuclear DNA run-on withaliquots of HeLa cells synchronized at the G1–S border and eitherreleased into S phase in the absence of drugs or maintained in thepresence of Aph and the replication checkpoint inhibitor 2-aminopurine (2-AP). 32P-labeled short nascent DNA, prepared atdifferent timepoints, was hybridized to a panel of DNA probesunique to the human rDNA repeats (supplementary material Fig.S7) to reveal the positions of replication forks. As previouslyreported (Balazs and Schildkraut, 1971), rDNA replication in HeLacells occurred throughout S phase; however, two peaks in earlyand middle S phase were detected in the present study (Fig. 5A,B).Analysis of the distribution of replication intermediates in thepresence of Aph, which restrains forks close to the replication

origins, produced surprising results (Fig. 5A,C). Initiation eventswere detected at similar frequencies throughout the rDNA unit incells arrested at the G1–S border or maintained for 3 hours in thepresence of Aph + 2-AP. Because this is the time when activeribosomal genes replicate (Figs 2–4), the random initiation patternis the opposite to what would be expected for the most highlytranscribed genomic loci on the basis of the model for therelationship between replication origin location and localtranscription (Mesner and Hamlin, 2005). It is unlikely that this isan artifact of the drug treatment, because site-specific replicationinitiation patterns in other transcribed loci are not altered under thesame conditions (Dimitrova and Gilbert, 2000). Similarly, theunexpected result cannot be explained by drug-induced alterationin transcription patterns, because neither chemical affected rDNAtranscription rates significantly (supplementary material Fig. S9).

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Fig. 5. Replication initiation and elongation patterns in the human rDNA repeats in the course of S phase. (A) 32P-labeled nuclear replication run-on productspurified from 106 HeLa cells synchronized at the G1–S border (0 Hrs) and either released into drug-free medium (No Drugs), or maintained in the presence of Aph+ 2-AP for the indicated times, were hybridized to a panel of human rDNA probes (1–18; see supplementary material Fig. S7) and a fragment of phage DNA ().DNA run-on (Exp) and RNA run-on hybridization results obtained with exponentially growing cells are shown as examples of random distribution of replicationforks or to visualize the transcribed portion of the rDNA unit, respectively. The schematic diagram of an rDNA repeat serves to illustrate probe allocation and is notto scale. The horizontal pins on the map mark EcoRI sites. The 18S and 28S rRNA coding regions are shown as dark boxes, and the start site and direction oftranscription are indicated by the arrow. (B) Replication rates in the human rDNA repeats during S phase. In cells released from the G1–S block in the absence ofdrugs, replication forks move unrestrained and asynchronously, and therefore are positioned randomly throughout the rDNA units. Relative c.p.m. values for allrDNA probes obtained by phosphoimaging of the ‘No Drugs, 0–12 Hrs’ blots shown in A were summed for each timepoint. Results were normalized to the valuesobtained for the G1–S-arrested cells and plotted against the elapsed time in S phase. (C) Replication initiation frequencies in the human rDNA repeats during Sphase. In cells maintained in the presence of Aph + 2-AP replication forks are arrested close to the replication origins. To correct for differences in probe size, basecomposition and hybridization efficiency, relative c.p.m. values obtained by phosphoimaging of the ‘Aph+2-AP, 3–12 Hrs’ blots in A were normalized to thecorresponding values for exponentially growing cells as described previously (Dimitrova and Gilbert, 1998) and plotted against the map position of each probe.The location of the 47S-rRNA coding region is indicated under the graph.

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Equally unexpected and even more striking is the focusing ofinitiation events to the non-transcribed intergenic spacers (NTSs)in cells maintained for 6–9 hours in the presence of Aph + 2-AP(i.e. the S-phase stage when the silent rRNA genes replicate).Notably, the peak of replication initiation activity coincided with apreferred initiation zone upstream of the rDNA promoter, whichhad also been identified in some previous studies (supplementarymaterial Fig. S7). These findings provide a potential explanationfor the contradictory results generated by earlier rDNA replicationorigin mapping endeavors (Little et al., 1993; Yoon et al., 1995;Gencheva et al., 1996; Lebofsky and Bensimon, 2005). It is possiblethat, owing to differences in experimental systems and design,and/or the capabilities of the methodologies involved, individuallabs preferentially detected one of the two, or a combination ofboth, initiation patterns specific for the two classes of ribosomalgenes.

RNA PolI-driven transcription is a determinant of thereplication initiation pattern in the human rRNA geneclustersI next asked whether the specific late S-phase initiation pattern isdependent on PolI-mediated transcription. Nuclear DNA run-onwas performed with aliquots of HeLa cells synchronized at theG1–S border and released into S phase for 6 or 9 hours in theabsence of drugs or maintained in the presence of Aph + 2-AP withor without the addition of 0.1 g/ml actinomycin D (AMD). AMDtreatment results in premature termination of rRNA transcripts.Even though at low doses (0.1 g/ml), some residual transcriptionof 5� external transcribed spacer (5�-ETS) leader sequences persists(supplementary material Fig. S10), as previously described formouse rDNA transcription (Fetherston et al., 1984; Puvion-Dutilleulet al., 1997), this AMD concentration was selected because itefficiently blocks transcription through most of the rDNA unit butwith little or no effect on PolII-directed transcription or DNAreplication (Baserga et al., 1965; Fetherston et al., 1984; Dimitrova,2006). The data presented in Fig. 6 show that selective inhibitionof rRNA synthesis resulted in drastic reduction of initiationspecificity in the mid and late-replicating rDNA copies, thusuncovering a role for PolI-mediated transcription in setting thereplication profile of human rDNA.

DiscussionThe lack of replicative activity inside mammalian nucleoli beforemid S phase (Nakamura et al., 1986; Nakayasu and Berezney,1989; O’Keefe et al., 1992; Dimitrova and Berezney, 2002) haspuzzled investigators for many years (Junera et al., 1995; Pliss etal., 2005), because the nucleoli are the residences of all expressedrRNA genes (Hadjiolov, 1985) and the latter are assumed toreplicate in early S phase (Little et al., 1993; Berger et al., 1997;Li et al., 2005). However, until recently, this assumption was basedsolely on the established general correlation between earlyreplication timing and the transcriptional activity of gene loci inhigher eukaryotes (Goren and Cedar, 2003), which, as is wellknown, does not apply to all transcribed loci (Taljanidisz et al.,1989; Farkash-Amar and Simon, 2010). Recent work with mousecells in the Grummt lab produced the first experimental evidencein support of this speculation (Li et al., 2005). The results describedhere lend further support to this concept through the use of humancells and a different experimental approach. Moreover, the findingsof the present report provide a clue to the location of the elusiveearly-replicating rDNA. I show that replicating rDNA is detectable

at the periphery of, or outside, nucleoli during the first half of Sphase in HeLa cells (Fig. 2A,B; supplementary material Fig. S5A;Table 1) and, following replication, it shifts back to the nucleolarinterior and reassociates with UBF (Fig. 4A; supplementarymaterial Fig. S6A), indicating that it mostly represents expressedribosomal gene copies. Notably, and contrary to the expectationsfor highly transcribed chromosomal loci (Mesner and Hamlin,2005), replication initiates randomly throughout the early-replicating rDNA repeats (Fig. 5), which probably represent activerDNA units. The data also show a markedly different spatiotemporalregulation of the silent rRNA genes. The latter replicate during midand late S phase, mostly in the nucleolar interior in replicationfactories assembled adjacent to active PolI transcription factories,

2749Dynamics of human rDNA replication

Fig. 6. PolI-mediated transcription sets the replication initiation pattern inthe human rDNA repeats during mid and late S phase. (A) 32P-labelednuclear replication run-on products purified from 106 HeLa cells synchronizedat the G1–S border (0 Hrs) and either released into drug-free medium(Control), or maintained in the presence of Aph + 2-AP alone or incombination with AMD, for 6 or 9 hours were hybridized to a panel of humanrDNA probes (1–18; see supplementary material Fig. S7) and a fragment ofphage DNA (). DNA run-on (Exp) and RNA run-on hybridization resultsobtained with exponentially growing cells are shown as examples of randomdistribution of replication forks or to highlight the transcribed segment of therDNA unit, respectively. (B) Relative c.p.m. values obtained byphosphoimaging of the blots in A were normalized as in Fig. 5 and plottedagainst the map position of each probe.

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with some rDNA replication also detectable at the nucleolarperiphery and outside nucleoli at this time. Unexpectedly,replication was found to initiate preferentially in the NTS of thelate-replicating rDNA repeats (Fig. 5) and PolI-driven transcriptionwas found to play a role in setting this specific initiation pattern(Fig. 6).

The behavior of the early-replicating rDNA reported here (Figs2 and 4; supplementary material Fig. S5A and Fig. S6A) mightprovide a rationale for the unexpected random initiation patternof the expressed (UBF-associated) rRNA genes. DNA replicationin mammalian cells is regulated largely by epigenetic mechanisms,and ongoing transcription is an essential determinant of thepreferred replication origin sites (Mesner and Hamlin, 2005;Dimitrova, 2006). When transcription is turned off, replicationinitiation sites redistribute along the chromatin fiber resulting inrandom initiation patterns (Dimitrova, 2006). It is thereforepossible that replication initiation complexes assembled in theNTSs of active rDNA units during G1 phase redistribute alongthe nucleosome-free rDNA when it departs from the intranucleolarPolI transcription factories to engage in the perinucleolarreplication factories. The organization of active and repressedribosomal genes in the human NORs has not been elucidated.However, the lack of a good correlation between the number ofrDNA copies on individual chromosomes and their transcriptionaloutput (de Capoa et al., 1988) is consistent with individualcompetent NORs hosting both active and silent rDNA copies.Furthermore, there is some evidence that active human rRNAgenes are clustered within competent NORs (Hamkalo and Miller,1973). Taken together with the lack of preferred initiation sites(Fig. 5), the relatively close origin spacing (Lebofsky andBensimon, 2005) and the inefficient function of the replicationfork barriers (RFBs) reported for human rDNA (Little et al.,1993; Lebofsky and Bensimon, 2005), this organization iscompatible with a model in which transcription is turned off inlarge clusters of active rRNA genes before their spatial relocationand replication. By contrast, unlike the active ribosomal genes,but similar to PolII-transcribed genomic loci (D.S.D. and P.Fraser, unpublished), the silent rDNA copies appear to retaintheir spatial positioning during transitions between transcriptionand replication, and they engage in intranucleolar replicationfactories assembled next to active PolI transcription factories. Inyeast NORs, replication always initiates in the NTS downstreamof active rRNA genes (Sogo and Thoma, 2004). As shown by thedata in Figs 5 and 6, a similar mechanism might operate inhuman rDNA at least for the silent rRNA genes located incompetent NORs. Interestingly, if the different replicationinitiation profiles uncovered here are conserved in all metazoans,these observations could account for the unexplained lack ofcorrespondence between the low proportion of transcriptionallyactive rDNA repeats and the higher than expected magnitude ofthe increase in replication origin specificity following activationof rDNA transcription during early development of Xenopusembryos (Hyrien et al., 1995).

Although unexpected, extranucleolar activity involving rDNAis not unprecedented. In yeast, recognition and initial processingof double-strand breaks (DSBs) in rDNA occur inside thenucleolus; however, the lesions are subsequently translocated inthe nucleoplasm where they are repaired by the recombinationmachinery. This transient relocation of DSBs in rDNA is essentialto prevent rDNA instability (Torres-Rosell et al., 2007). By asimilar logic, it is possible that replicating the active rRNA genes

at the periphery, or outside, nucleoli reduces the risk of collisionsbetween the transcription and replication machineries that cancause DSBs and promote rDNA hyper-recombination. Theribosomal genes are the most highly transcribed genomic loci,with 100–150 PolI molecules loaded onto each active gene(Hamkalo and Miller, 1973), and this represents a serious obstaclefor their replication. The existence of a robust mechanism,independent of the RFBs, which coordinates the activity oftranscription and replication complexes in the competent NORsis implied by the bidirectionality and low efficiency of RFBs inhuman rDNA (Lebofsky and Bensimon, 2005). Furthermore,replicating the active rDNA copies at the periphery of, or outside,the nucleolar compartment in the absence of transcriptionalinterference might provide an additional advantage by promotingmore efficient rDNA replication, reminiscent of the moresynchronous activation of replicons and the higher replicationrates of heterochromatin (Comings, 1970). Streamlinedreplication, on its part, will enable faster switchback to theessential growth-supporting function of the ribosomal genes,namely production of structural rRNAs for building ribosomes(Hadjiolov, 1985).

Materials and MethodsCell culture and synchronizationHeLa cells were propagated in Dulbecco’s modified Eagle’s medium (Invitrogen)supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in a 5% CO2

atmosphere. Cell populations synchronized in mitosis or at the G1–S border wereprepared as described previously (Dimitrova and Gilbert, 1998; Dimitrova andGilbert, 1999; Dimitrova and Gilbert, 2000).

For the replication run-on experiments, parallel cultures of HeLa cells, arrestedwith aphidicolin at the G1–S border, were either released in drug-free medium ormaintained in the presence of aphidicolin (Sigma; 50 g/ml) and 2-aminopurine(Sigma; 10 mM) alone, or in combination with actinomycin D (Sigma; 0.1 g/ml).

Visualization of replication foci, transcription foci and nuclear proteinsPulse-labeling of nascent DNA with 5-bromo-2�-deoxyuridine (BrdU; Sigma) andits detection by indirect immunofluorescence were performed as described previously(Dimitrova and Gilbert, 1999; Dimitrova and Gilbert, 2000; Dimitrova, 2006;Dimitrova, 2009). For direct detection of nascent RNA and DNA in situ, HeLa cellsgrown on coverslips were pulse-labeled for 10 minutes by supplementing the cellculture medium with 100 M 5-ethynyl uridine (EU; Invitrogen) or 50 M 5-ethynyl-2�-deoxyuridine (EdU; Invitrogen), respectively. The cells were fixed for 10minutes with 4% formaldehyde in PBS and permeabilized with 0.5% Triton X-100in PBS for 10 minutes at room temperature. EU and EdU were stained with AlexaFluor azide derivatives (Invitrogen) through click chemistry reactions broadlyfollowing the manufacturer’s recommendations (Dimitrova, 2009). UBF and nucleolinwere detected with a rabbit polyclonal antibody (Sigma) or a mouse monoclonalantibody (Santa Cruz Biotechnology), respectively, and appropriate Alexa-Fluor-conjugated secondary antibodies (Invitrogen).

3D DNA FISHHeLa cells grown on coverslips were fixed for 10 minutes with 4% formaldehydein PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 minutes at roomtemperature and then incubated with RNase A (Sigma; 200–300 g/ml in PBS) for2–4 hours at 37°C. DNA FISH was performed as described previously (van Dekkenet al., 1988). No FISH signals were detected when digestion with exonuclease IIIwas omitted. For detection of rDNA in situ, EcoRI fragments A (7.3 kb) and B (5.8kb) (supplementary material Fig. S7) (Sylvester et al., 2004) were labeled withdigoxigenin-11-2�-deoxyuridine-5�-triphosphate or biotin-11-2�-deoxyuridine-5�-triphosphate by standard nick-translation and detected with sheep anti-digoxigeninantibody (Roche) and 1–2 layers of Alexa-Fluor-conjugated secondary antibodies(Invitrogen), or with Alexa-Fluor-conjugated streptavidin (Invitrogen), respectively.

Laser scanning confocal microscopyStacks of serial optical sections (0.25–0.5 m apart) were collected through theHeLa nuclei sequentially for 3–4 fluorochromes using a Zeiss LSM 510 Metasystem housed on an inverted Axiovert 200 microscope equipped with a Plan-Apo63�1.4 NA oil-immersion objective. The LSM Image Browser (Carl Zeiss) wasused to analyze the three-dimensional stacks without further processing. Line-scanplots of fluorescence intensity versus distance were generated from selected opticalsections using the RGB Profiler Plugin in ImageJ 1.39. Graphs were created using

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DeltaGraph 5 for the Macintosh (Red Rock Software). Figures were assembled fromindividual images using Adobe Photoshop CS4 and Adobe Illustrator CS4 software.

Nuclear transcription and replication run-on assaysConditions for nuclear RNA and DNA run-on analysis were as described previously(Dimitrova and Gilbert, 2000; Dimitrova, 2006). Briefly, transcription run-on reactionswere performed at 21°C in transcription cocktail supplemented with 25–100 Ci [-32P]UTP (NEN). For measurements of global transcription rates (2.5�105 nuclei persample), aliquots were removed at various times thereafter and the amount of[32P]UTP incorporated into RNA was determined by acid precipitation. For analysisof rDNA transcription, 1�106 nuclei were used per sample and incubated for 30minutes at 21°C. 32P-labeled RNA was purified and hybridized to a panel of rDNAprobes immobilized on nylon membranes (Hybond N+, Amersham-GE Healthcare;1 g DNA per slot). All probes (size range 250–2200 bp) were tested by Southernblot analysis for their uniqueness to rDNA and a lack of highly repetitive sequences.A fragment of phage DNA was used as a negative control for non-specifichybridization.

In the rDNA replication analysis, for each experimental condition, nuclei wereprepared from 1�106 cells and incubated for 30 minutes at 21°C in replicationcocktail supplemented with 100 Ci [-32P]-dATP (NEN) to label nascent DNA atexisting replication forks. 32P-labeled nuclear replication run-on products werepurified and hybridized to the same panel of human rDNA probes.

To correct for differences in probe size, base composition and hybridizationefficiency, relative c.p.m. for each probe obtained by phosphoimaging of the blots(Molecular Dynamics) were normalized to the corresponding values for exponentiallygrowing cells as described previously (Dimitrova and Gilbert, 1998; Dimitrova andGilbert, 2000; Dimitrova, 2006).

This paper is dedicated to the memory of Asen Hadjiolov. I amgrateful to J. Silvester for the provision of human rDNA clones, andto P. Fraser for the use of laboratory space and reagents. This workwas supported by the Biotechnology and Biological Sciences ResearchCouncil, UK.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/16/2743/DC1

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